What are the mechanisms of T cell-mediated tumor regression? Understanding the answers to this question is of critical importance, since it should provide opportunities to monitor, refine and improve immunotherapy trials. Direct cytotoxicity has long been implicated in T cell-mediated tumor regression. However, we recently reported that the adoptive transfer of effector T cells from perforin k/o (PKO) or FasL deficient (gld) mice results in regression of B16BL6-D5 (D5) melanoma and cures animals with systemic tumor (3). Additional studies with mice deficient in both IFN-gamma and perforin (PIG) also mediate tumor regression and we have developed 2 lines of mice deficient in 3 critical effector mechanisms to address the question of "what mechanism(s) is essential for T cell-mediated tumor regression?" A related question is "what death pathway(s) is responsible for T cell-mediated tumor cell destruction?" Is it through a caspase 8, caspase 9 or a caspase- independent process? Aims 2 and 3 will address this question. Identification of the critical death pathway(s) could lead to determination of mechanisms and allow implementation of novel approaches to augment efficacy. Aims 4 and 5 exploit these advances and will test whether an innovative strategy, that we have developed, will amplify T cell-mediated tumor destruction by amplifying the death pathways initiated by CTL (including granzyme B and the TNF family members). A series of novel pro-drug molecules have been designed that can be delivered to tumors and will only be activated once a T cell initiates a death pathway. These could overcome the inhibitors of apoptosis that have been identified in many tumors and which may be responsible for the failure of immunotherapy strategies. If this innovative approach using novel immune activated pro-drug molecules is successful in these preclinical studies it could be rapidly translated into phase I clinical trials for patients with cancer.